These TRE-hM3Dq transgenic mice may be bred to generate Tet-Off/Tet-On mutant mice with conditional (inducible/reversible) expression of hM3Dq; a mutant G protein-coupled receptor (GPCR) that activates the canonical Gq pathway specifically following administration of the pharmacologically inert molecule clozapine-N-oxide (CNO). TRE-hM3Dq transgenic mice may be useful for chemogenetic/pharmacogenetic applications to study receptor-specific functions or general downstream cellular signaling emanating from the activated GPCR.
Bryan L Roth, University of North Carolina at Chapel Hill
Hemizygous TRE-hM3Dq transgenic mice are viable and fertile, with no reported phenotypic abnormalities. The TRE-hM3Dq transgene has a modified Tet response element (TRE or tetO) upstream of the mutant G protein-coupled receptor, hM3Dq (a human muscarinic 3 receptor with two amino acid substitutions (Y149C3.33/A239G5.46) that abolish receptor affinity for the native ligand, acetylcholine (ACh), but allow receptor binding and subsequent activation by the small pharmacologically inert molecule clozapine-N-oxide (CNO)). When bred with another mouse expressing tetracycline-controlled transactivator protein (tTA) or reverse tetracycline-controlled transactivator protein (rtTA), hM3Dq expression in the resulting double mutant offspring can be regulated with tetracycline or its analog doxycycline (dox). As designed, TRE-hM3Dq transgenic mice have no reported levels of hM3Dq activity in tTA(+dox) or rtTA(-dox) cell types. In TRE-hM3Dq transgenic tTA(-dox) or TRE-hM3Dq transgenic rtTA(+dox) cells types, hM3Dq expression results in activation of the canonical Gq pathway only following administration of CNO.
These TRE-hM3Dq transgenic mice may be useful to study receptor-specific functions or general downstream cellular signaling emanating from the activated G protein-coupled receptor (GPCR). For example, when bred to a strain expressing tTA in forebrain neurons (Camk2a-tTA; see Stock No. 003010), these transgenic mice are a model allowing in vivo chemical control of neuronal activity, neuronal firing, and non-neuronal signaling.
In addition, breeding TRE-hM3Dq mice with cfos-htTA mice (Stock No. 018306) generates double transgenic offspring with hM3Dq expression in excitatory neurons that are sufficiently active to drive the c-fos promoter. Those hM3Dqfos double transgenic mice allow CNO ligand-induced, artificial reactivation of neurons associated with a specific memory. Temporal modulation may also be employed using dox. Such hM3Dqfos double transgenic mice allow one to generate and artificially-activate a synthetic memory trace, and may be useful in studying the spatial pattern of activity at the time of learning and retrieval.
Of note, several chemogenetic/pharmacogenetic tool strains are available from The Jackson Laboratory Repository; including these Tet-responsive TRE-hM3Dq transgenic mice (Stock No. 014093), the adora2A-rM3Ds transgenic mice (Stock No. 017863), the Tet-responsive TRE-hM4Di transgenic mice (Stock No. 024114), the Cre-inducible R26-LSL-Gi-DREADD mice (R26-hM4Di/mCitrine; Stock No. 026219), the Cre-inducible R26-LSL-Gq-DREADD mice (R26-hM3Dq/mCitrine; Stock No. 026220) and the Cre-inducible R26-LSL-Gs-DREADD mice (R26-rM3Ds/mCitrine; Stock No. 026261).
For CNO protocol, see the detailed protocol for dissolving CNO obtained from the attachments section of the Designer Receptors Exclusively Activated by Designer Drugs DREADD wiki webpage.
The TRE-HA-hM3Dq transgene was designed with a modified Tet response element (TRE or (tetO); described in detail below), a hemagglutinin epitope tag (HA), the hM3Dq sequence, and an SV40 polyA signal. The hM3Dq sequence is a Gq-coupled human M3 muscarinic DREADD (designer receptor exclusively activated by designer drug). To create the hM3Dq sequence, the wildtype human muscarinic 3 receptor (CHRM3) sequence was modified via site-directed mutagenesis to harbor two amino acid substitutions (Y149C3.33/A239G5.46) that abolish receptor affinity for the native ligand, acetylcholine (ACh), but allow receptor binding and subsequent activation by the small drug-like molecule clozapine-N-oxide (CNO).
This 2.36 kb TRE-HA-hM3Dq transgene was injected into the pronuclei of B6SJL hybrid mouse oocytes. Founder TRE-hM3Dq mice (line 1) were bred with B6;CBA-Tg(Camk2a-tTA)1Mmay/J (see Stock No. 003010) to establish the colony. TRE-hM3Dq transgenic mice were bred together or to wildtype (noncarrier) mice from the colony for approximately eight generations, and then crossed with C57BL/6J mice for one or two generations. The Camk2a-tTA transgene was removed during these breedings. Upon arrival at The Jackson Laboratory Repository, TRE-hM3Dq transgenic mice were bred to C57BL/6J inbred mice (Stock No. 000664) for at least one generation to establish the colony.
Additional information on hM3Dq:
More information on hM3Dq or other DREADDs may be available at the University of North Carolina Designer Receptors Exclusively Activated by Designer Drugs DREADD wiki webpage.
Additional information on TRE promoter:
The TRE promoter used in these transgenic mice is the Tet-responsive Ptight promoter. This Ptight promoter contains a modified Tet response element (TREmod), which consists of seven direct repeats of a 36-bp sequence each containing the 19-bp tet operator sequence (tetO). The TREmod is just upstream of a minimal human cytomegalovirus (CMV) promoter (PminCMVΔ), which lacks the enhancer that is part of the complete CMV promoter. Consequently, Ptight is silent in the absence of binding of TetR or rTetR to the tetO sequences.
|Expressed Gene||CHRM3, cholinergic receptor, muscarinic 3, human|
|Site of Expression|
|Allele Name||transgene insertion 1, Bryan Roth|
|Allele Type||Transgenic (Inducible, Inserted expressed sequence)|
|Allele Synonym(s)||TRE-HA-hM3Dq; TRE-hM3Dq; tetO-hM3Dq|
|Gene Symbol and Name||Tg(tetO-CHRM3*)1Blr, transgene insertion 1, Bryan Roth|
|Promoter||CMV, cytomegalovirus, human|
|Promoter||tetO, tet operator,|
|Expressed Gene||CHRM3, cholinergic receptor, muscarinic 3, human|
|Strain of Origin||(C57BL/6 x SJL)F1|
|Molecular Note||The Tet-responsive P promoter which consists of seven direct repeats of 36-bp sequence each containing the 19-bp tet operator sequence (tetO) is just upstream of a minimal human cytomegalovirus (CMV) promoter. This is upstream of a hemagglutinin epitoptag and a Gq-coupled human M3 muscarinic receptor. The wildtype human muscarinic 3 receptor sequence was modified via site-directed mutagenesis to harbor two amino acid substitutions (Y149C and A239G) that abolish receptor affinity for the native ligand,acetylcholine (ACh), but allow receptor binding and subsequent activation by the small drug-like molecule clozapine-N-oxide (CNO).|
|Mutations Made By|| |
Bryan Roth, University of North Carolina at Chapel Hill
When maintaining a live colony, transgene carrier mice may be bred together or to wildtype (noncarrier) mice from the colony. The donating investigator reports they have not tried to generate homozygous mice (November 2011).
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